GRK2 promotes activation of lung fibroblast cells and contributes to pathogenesis of pulmonary fibrosis through increasing Smad3 expression.

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor-ß1 (TGF-ß1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-ß1-induced signal transduction in different cell types; however, the role of GRK2 in TGF-ß1-induced activation of lung fibroblast cells and development of pulmonary fibrosis has not been revealed. In this study, we found that GRK2 levels were increased in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-ß1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Posttreatment with GRK2 inhibitor reduced extracellular matrix (ECM) accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-ß1-induced lung fibroblast cells or myofibroblast cells isolated from patients with pulmonary fibrosis. Furthermore, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-ß1 signal pathway and ECM accumulation.

Resveratrol ameliorates myocardial fibrosis by regulating Sirt1/Smad3 deacetylation pathway in rat model with dilated cardiomyopathy.

BACKGROUND: The aim of this study was to investigate the effects of Resveratrol (RSV) in rats with dilated cardiomyopathy (DCM). METHODS: Porcine cardiac myosin was used to set up rat model with DCM. RSV (10 mg/kg in RSV-L group and 50 mg/kg in RSV-H group) or vehicle was administered to rats with DCM once daily from the 28th day till the 90th day after the first immunization. Cardiac function of rats was evaluated by echocardiographic analysis. The deposition of fibrous tissues in the hearts was evaluated by Masson and picrosirius red staining. The mRNA levels of collagen type I (Col I), collagen type III (Col III) and silence information regulator 1 (Sirt1) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction of Sirt1 with Smad3 was revealed by coimmunoprecipitation. RESULTS: The heart weight, heart weight/body weight ratio, left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were significantly increased in rats with DCM, and attenuated by RSV. RSV also positively decreased fibrosis, and the expression of Col I and Col III in the myocardium. The Sirt1 mRNA was significantly decreased in myosin-immunized hearts and was positively increased by RSV. The Sirt1 combined with Smad3 directly. Acetylation of Smad3 (Ac-Smad3) was significantly increased in DCM and was markedly decreased by RSV. CONCLUSION: RSV effectively ameliorated myocardial fibrosis and improved cardiac function by regulating Sirt1/Smad3 deacetylation pathway in rat model with DCM.

Specific Smad2/3 Linker Phosphorylation Indicates Esophageal Non-neoplastic and Neoplastic Stem-Like Cells and Neoplastic Development.

BACKGROUND: There is little known about stem cells in human non-neoplastic and neoplastic esophageal epithelia. We have demonstrated expression of linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr), suggesting presence of stem-like cells in mouse esophageal epithelium, and identified presence of pSmad2/3L-Thr-positive cells that might function as cancer stem cells in mouse model of colorectal carcinoma. AIMS: We explore whether pSmad2/3L-Thr can be used as a biomarker for stem cells of human esophageal epithelia and/or neoplasms. METHODS: We have used esophageal tissues from inpatients undergoing endoscopic submucosal dissection and performed double immunofluorescent staining of pSmad2/3L-Thr and Ki67, CDK4, p63, Sox2, CK14, p53, ALDH1, CD44 or D2-40 after which the sections were stained with hematoxylin and eosin. RESULTS: pSmad2/3L-Thr-positive cells showed immunohistochemical co-localization with CDK4, p63, CD44 and Sox2 in the basal and parabasal layers of non-neoplastic esophageal epithelia. In esophageal neoplasms, they showed immunohistochemical co-localization with p53, CDK4, ALDH1 and CD44. There was a significant increase in the percentage of pSmad2/3L-Thr-positive cells in the p53-positive neoplastic cell population with development of esophageal neoplasia. pSmad2/3L-Thr-positive cells localized to the lower section of low-grade intraepithelial neoplasia and were observed up to the upper section in carcinoma in situ. In invasive squamous cell carcinoma, they were scattered throughout the tumor with disappearance of polarity and were found in intraepithelial primary lesions and sites of submucosal and vessel invasion. CONCLUSIONS: We determined significant expression of pSmad2/3L-Thr in human esophageal non-neoplastic and neoplastic epithelia, indicating that these are epithelial stem-like cells and cancer stem cells, respectively, that correlate with developing esophageal neoplasms.

Glycyrrhizic acid, as an inhibitor of HMGB1, alleviates bleomycin-induced pulmonary toxicity in mice through the MAPK and Smad3 pathways.

AIM: High-mobility group box 1 (HMGB1) protein has been noticed particularly for its pivotal role in several pathologies. However, the relevance between HMGB1 and pathological progress in lung toxicity still remains unclear. In the study, we evaluated the effect of glycyrrhizic acid as an HMGB1 inhibitor on the early inflammation and late fibrosis in bleomycin-induced pulmonary toxicity in mice. METHODS: We established a bleomycin-induced pulmonary toxicity model to detect the relevance between HMGB1 and pathological changes in the early inflammatory and late fibrotic stages. RESULTS: We found that bleomycin-induced increase in inflammatory cytokines interleukin (IL)-ß1, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and inflammatory lesions in lung tissue in the early stage of the model. However, markers of fibrosis such as transforming growth factor (TGF)-ß1 and α-smooth muscle actin (α-SMA) were significantly elevated on day 7 after bleomycin instillation. Interestingly, HMGB1 also began to rise on day 7, rather than in the early inflammatory phase. However, early (from day 0 to 14 after bleomycin instillation) or late (from day 14 to 28) intervention with HMGB1 neutralizing antibody or glycyrrhizic acid alleviated inflammation and fibrosis through down-regulating the inflammatory signaling mitogen-activated protein kinase (MAPK) and fibrotic signaling Smad3 pathway. CONCLUSION: Our results suggested that HMGB1 mediates both inflammation and fibrosis in this model. The development of high-potency and low-toxicity HMGB1 inhibitors may be a class of potential drugs for the treatment of pulmonary fibrosis.

TGF-ß1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-ß1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH). METHODS: Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (•OH) and oxidative DNA damage were measured using an •OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. RESULTS: In culture, TGF-ß1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H2O2-triggered •OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct •OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions +2-+13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-ß1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA+pSMAD2+- and αSMA+NOX4+-positive hepatic stellate cells from patients with NASH and advanced fibrosis. CONCLUSIONS: Downregulation of CYGB by the TGF-ß1/pSMAD2/SP3-M1 pathway brings about •OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH. LAY SUMMARY: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-ß1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-ß. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Lactic Acid Downregulates Viral MicroRNA To Promote Epstein-Barr Virus-Immortalized B Lymphoblastic Cell Adhesion and Growth.

High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B-cell lymphoma. Here, we report that lactate dehydrogenase A (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA; acidic lactate) at low concentration triggers EBV-infected B-cell adhesion, morphological changes, and proliferation in vitro and in vivo Moreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III, and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression of SMAD3, JUN, and COL1A genes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment.IMPORTANCE The tumor microenvironment is complicated, and lactate, which is created by cell metabolism, contributes to an acidic microenvironment that facilitates cancer progression. However, how LA operates in virus-associated cancers is unclear. Thus, we studied how EBV (the first tumor virus identified in humans; it is associated with many cancers) upregulates the expression of LDH-A and lactate production in B lymphoma cells. Elevated LA induces adhesion and the growth of EBV-infected B cells by inhibiting viral microRNA transcription. Thus, we offer a novel understanding of how EBV utilizes an acidic microenvironment to promote cancer development.

Tenofovir alafenamide fumarate attenuates bleomycin-induced pulmonary fibrosis by upregulating the NS5ATP9 and TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Pulmonary fibrosis is a progressive and irreversible disease for which therapeutic options are currently limited. A recent in vivo study showed that tenofovir, a nucleotide analogue reverse transcriptase inhibitor, had direct antifibrotic effects on skin and liver fibrosis. Another study in vitro revealed that NS5ATP9 inhibited the activation of human hepatic stellate cells. Because of the similarity of fibrotic diseases, we hypothesized that tenofovir alafenamide fumarate (TAF), the prodrug of tenofovir, and NS5ATP9, is related to and plays a role in the suppression of pulmonary fibrosis. METHODS: We investigated the influence of NS5ATP9 on fibrosis in vitro. Human lung fibroblasts (HFL1) were transfected with short interfering RNAs or overexpression plasmids of NS5ATP9 before stimulation by human recombinant transforming growth factor-ß1. The effect of TAF was evaluated in a bleomycin-induced fibrosis murine model. Male C57BL/6 mice were treated with bleomycin on day 0 by intratracheal injection and intragastrically administered TAF or vehicle. Left lung sections were fixed for histological analysis, while homogenates of the right lung sections and HFL1 cells were analyzed by western blotting and quantitative reverse transcription polymerase chain reaction. RESULTS: NS5ATP9 suppressed the activation of lung fibroblasts. Upregulation of collagen type 3 (α 1 chain) and α-smooth muscle actin was observed in HFL1 cells when NS5ATP9 was silenced, and vice-versa. TAF also showed anti-fibrotic effects in mice, as demonstrated by histological analysis of fibrosis and expression of extracellular matrix components in the lung sections. Additionally, TAF inhibited transforming growth factor-ß1 and phosphorylated-Smad3 synthesis in HFL1 cells and the murine model, which was accompanied by upregulation of NS5ATP9. CONCLUSIONS: Our results suggest that NS5ATP9 forms a negative feedback pathway in pulmonary fibrosis and TAF has anti-fibrotic properties as it upregulates the expression level of NS5ATP9. As TAF has been shown to be safe and well-tolerated in humans, TAF and NS5ATP9 may be useful for developing novel therapeutics for pulmonary fibrosis.

Schizandrin protects H9c2 cells against lipopolysaccharide-induced injury by downregulating Smad3.

Schizandrin is a major bioactive constituent of Schisandra chinensis (Turcz.) Baill with antioxidant and anti-inflammatory properties. The objective of this study was to explore the potential effects of schizandrin on a cell model of myocarditis. The H9c2 cells were treated with schizandrin alone or in combination with lipopolysaccharide (LPS), after which, cell survival, migration, and the release of inflammatory cytokines were assessed. Moreover, downstream effectors and signaling pathways were studied to reveal the possible underlying mechanism. As a result, LPS stimulation induced significant cell damage as cell viability was repressed and the apoptosis was induced. In the meantime, LPS promoted the release of proinflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL-6, and tumor necrosis factor (TNF-α) while repressing the release of the anti-inflammatory cytokine IL-10. Schizandrin could promote H9c2 cell migration and long-term treatment (7 days) enhanced cell viability. More interestingly, pretreatment with schizandrin attenuated LPS-induced cell loss and inflammatory response. Besides this, Smad3 was a downstream effector of schizandrin. The beneficial effects of schizandrin on the H9c2 cells were attenuated when Smad3 was overexpressed. Moreover, the silencing of Smad3 deactivated c-Jun N-terminal kinase (JNK) and nuclear factor κB (NF-κB) pathways. This study preliminarily demonstrated that schizandrin prevented LPS-induced injury in the H9c2 cells and promoted the recovery of myocardial tissues by enhancing cell viability and migration. Schizandrin conferred its beneficial effects possibly by downregulating Smad3 and inhibiting the activation of JNK and NF-κB pathways.

Curcumin mitigates the epithelial-to-mesenchymal transition in biliary epithelial cells through upregulating CD109 expression.

Biliary epithelial cells (BECs) can secrete bile and the epithelial-to-mesenchymal transition (EMT) of BECs can cause fibrosis or damage interlobular bile ducts, leading to chronic cholangiopathies, such as primary biliary cholangitis (PBC). Transforming growth factor-ß1 (TGF-ß1) is a potent inducer of the EMT while curcumin, a diarylheptanoid, can inhibit the EMT of hepatocytes in many liver diseases. However, the protection and underlying mechanisms of curcumin against the EMT of BECs have not been clarified. Herein, we show that curcumin treatment significantly mitigates the EMT of BECs in vitro and in vivo. Mechanistically, curcumin significantly attenuated the TGF-ß1-induced Smad and Hedgehog signaling, and upregulated CD109 expression in BECs. Collectively, these findings highlighted the therapeutic potential of curcumin to counteract the EMT process in PBC.

TGF-ß Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways.

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF-ß) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF-ß induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF-ß. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF-ß in a Smad3-independent manner. Collectively, our data suggest that TGF-ß stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566-579, 2018. © 2017 Wiley Periodicals, Inc.

Investigating the mechanism by which SMAD3 induces PAX6 transcription to promote the development of non-small cell lung cancer.

BACKGROUND: This study investigated the function of SMAD3 (SMAD family member 3) in regulating PAX6 (paired box 6) in non-small cell lung cancer. METHODS: First, qRT-PCR was employed to detect SMAD3 expression in cancer tissues along with normal tissues and four cell lines, including BEAS-2B, H125, HCC827 and A549 cells. SMAD3 was knocked down by small interference RNA (siRNA), and then its expression was determined via qRT-PCR and Western blot analysis. The correlation between SMAD3 and PAX6 was determined by double luciferase reporter experiments and chromatin immunoprecipitation (ChIP) assay. Cell viability was evaluated by CCK-8 and colony forming assays, while cell migration and invasion were detected by Transwell analysis. RESULTS: SMAD3 and PAX6 were upregulated in lung cancer tissues and cancer cells. Knocking down SMAD3 and PAX6 by transfection with siRNAs specifically suppressed the expression of SMAD3 and PAX6 mRNA and protein levels. SMAD3 could promote PAX6 transcriptional activity by binding to its promoter. Reduced expression of SMAD3 led to the downregulation of PAX6 mRNA and protein levels along with decreased cell migration, invasion, proliferation and viability in A549 and HCC827 cells. PAX6 overexpression altered the si-SMAD3-induced inhibition of cell migration, invasion, proliferation and viability in A549 and HCC827 cells. Additionally, PAX6 knockdown alone also repressed the cell migration, invasion, proliferation and viability of the cell lines. CONCLUSIONS: SMAD3 promotes the progression of non-small cell lung cancer by upregulating PAX6 expression.

Identification of a novel intergenic miRNA located between the human DDC and COBL genes with a potential function in cell cycle arrest.

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.

TGF-ß1 and connexin-43 expression in neurogenic bladder from rats with sacral spinal cord injury.

AIMS: Sacral spinal cord injury (SCI) could induce underactive bladder (UAB). Malfunction of connexin 43 (CX43) regulated by TGF-ß1 might involve in urinary bladder dysfunction. We studied the changes of CX43 and TGF-ß1/Smad3 signaling in detrusor of neurogenic bladder (NB) in sacral SCI rats. METHODS: Sacral SCI was produced by hemisection (SSCH) or transection (SSCT) of spinal cord between L4 and L5 in female Wistar rats. BBB scores, residual urine volume and bladder weight as well as characteristic cystometric parameters at 6th week were used to confirm the successful establishment of NB. Western blotting and qRT-PCR were used to exam the protein and mRNA expression levels of CX43, CX45, TGF-ß1, and Smad3 in detrusor. RESULTS: BBB scores were significantly decreased, with the lowest in SSCT rats (P < 0.01). The residual urine volume, mean bladder weight, and cystometric parameters were increased, with the highest in SSCT rats. CX43 and phospho-CX43 protein levels were significantly decreased, but those of TGF-ß1, Smad3, and phospho-Smad3 were significantly increased. It was the protein and mRNA levels of CX43 but not those of CX45 which were decreased in negative accordance with those of TGF-ß1 and Smad3. Those changes were more significant in SSCT than in SSCH rats. CONCLUSIONS: This study indicates that voiding dysfunction is related to the decreased CX43 function in detrusor from NB. TGF-ß1/Smad3 signaling might be involved in the down-regulation of CX43 in SCI rats. Early regulation of CX43 might be beneficial to patients with voiding dysfunction.

All-Trans Retinoic Acid supplementation prevents cardiac fibrosis and cytokines induced by Methylglyoxal.

Methylglyoxal (MG), a metabolic intermediate of glycolysis is a precursor for endogeneous production of advanced glycation end-products. The increased production of MG have negative influence over the structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic cardiac complications. Retinoic acid (RA), an active metabolite of vitamin A, has a major role in preventing cardiac remodeling and ventricular fibrosis. Hence, the objective of the present study was to determine whether rats administered with all-trans retinoic acid (RA) could attenuate MG induced pathological effects. Wistar rats were divided into 4 groups. Group 1 rats were kept as control; Group 2 rats were administrated with MG (75 mg/kg/day) for 8 weeks. Group 3 rats were given RA (Orally, 1.0 mg/kg/day) along with MG; Group 4 rats received RA alone. Cardiac antioxidant status, induction of fibrosis, AGE receptor (RAGE) and cytokines expression was evaluated in the heart tissues. Administration of MG led to depletion of antioxidant enzymes, induction of fibrosis (p < 0.001), up-regulated expression of RAGE (3.5 fold), TGF-ß (4.4 fold), SMAD2 (3.7 fold), SMAD3 (6.0 fold), IL-6 (4.3 fold) and TNF-α (5.5 fold) in the heart tissues compared to control rats. Moreover, the exogenous administration of MG caused significant (p < 0.001) increase in the circulating CML levels. Whereas, RA treatment prevented the induction of fibrosis and restored the levels of cytokines and RAGE expression. Methylglyoxal-induced fibrosis can lead to pathological effects in the heart tissues. RA attenuates the effects of MG in the heart, suggesting that it can be of added value to usual diabetic therapy.

Rotavirus-induced miR-142-5p elicits proviral milieu by targeting non-canonical transforming growth factor beta signalling and apoptosis in cells.

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa-miR-142-5p was up-regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR-142-5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR-142-5p. Functional studies of hsa-miR-142-5p identified its role in transforming growth factor beta (TGFß) signalling as TGFß receptor 2 and SMAD3 were degraded during both hsa-miR-142-5p overexpression and rotavirus infection. TGFß is induced during rotavirus infection, which may promote apoptosis by activation of non-canonical pathways in HT29 cells. However, up-regulated miR-142-5p resulted in the inhibition of TGFß-induced apoptosis suggesting its anti-apoptotic function. Rotavirus NSP5 was identified as a regulator of miR-142-5p expression. Concurrently, NSP5-HT29 cells showed inhibition of TGFß-induced apoptosis and epithelial to mesenchymal transition by blocking non-canonical pathways. Overall, the study identified proviral function of hsa-miR-142-5p during rotavirus infection. In addition, modulation of TGFß-induced non-canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.

CXCL9 Regulates TGF-ß1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-ß1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-ß1-induced EMT. A decrease in TGF-ß1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-ß1-induced EMT.

Airway smooth muscle cells from ovalbumin-sensitized mice show increased proliferative response to TGFß1 due to upregulation of Smad3 and TGFßRII.

OBJECTIVE: This study aimed to elucidate the role of Transforming growth factor (TGF)-ß1 signaling in the proliferation of airway smooth muscle cells (ASMCs). BACKGROUND: TGF-ß1 is an important cytokine in airway remodeling in asthma. However, results of studies focusing on the effect of TGFß1 on proliferation of ASMCs are controversial. METHODS: An allergic model that mimics airway remodeling in chronic asthma was established and primary ASMCs were cultured. Cell proliferation was detected by viable cell counting and Cell Counting Kit (CCK)-8 analysis. Expression and phosphorylation of Smad3, type 1 TGFß receptor (TGFßRI), type 2 TGFß receptor (TGFßRII), extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), C-Jun N-terminal kinase (JNK) and AKT were detected by western blot. siRNAs were used to knock down Smad3 and TGFßRII. RESULTS: Smad3 and TGFßRII were up-regulated in primary ASMCs isolated from ovalbumin (OVA)-sensitized mice as compared with ASMCs isolated from unsensitized control mice, which persisted for at least four passages. TGFß1 stimulated proliferation of ASMCs isolated from OVA-sensitized mice, which was inhibited by specific siRNA targeting Smad3 or TGFßRII. However ASMCs from control mice showed no proliferative response to TGFß1. TGFß1-induced proliferation of ASMCs from OVA-sensitized mice was markedly attenuated by PD-98059, a specific ERK1/2 inhibitor. TGFß1 induced ERK1/2 phosphorylation within 15 minute, which was partially blocked by specific inhibitor of Smad3 (SIS3). CONCLUSIONS: ASMCs isolated from OVA-sensitized mice showed hyper-proliferation upon TGFß1 stimulation. This might have been associated with up-regulated Smad3 and TGFßRII and mediated by ERK1/2 downstream to Smad3.

TGFß1-Smad3-Jagged1-Notch1-Slug signaling pathway takes part in tumorigenesis and progress of tongue squamous cell carcinoma.

BACKGROUND: TGFß1 and Smad3 play an important role in the process of EMT. TGFß1 regulates the expression of Jagged1 by modulating Notch signaling. Jagged1 is related to tumor invasion, metastasis, chemotherapy resistance, and tumor immune escape. The aims of this study are to examine deregulation of TGFß1-Smad3-Jagged1-Notch1-Slug signaling in TSCC and to investigate its roles in TSCC progression. MATERIALS AND METHODS: Notch1, Smad3, Jagged1 and Slug proteins and mRNA expression were detected in specimens from 89 cases of patients. We analyzed the correlation between their expressions and histological grade, clinical stage and lymph node metastasis. RESULTS: Notch1, Smad3, Jagged1 and Slug mRNA expressions in TSCC were higher than normal tissue (P <0.05). The protein expression of Notch1 and Smad3 in TSCC were higher (χ(2) =7.30, P <0.01 and χ(2) = 5.84, P <0.05). Notch1 and Smad3 expressions were correlated with clinical stage (χ(2) =18.81, P<0.01; χ(2) =22.29, P<0.01), but not Jagged1 (χ(2) =0.53, P>0.05). The Slug protein expression was correlated with clinical stage. The positive rate of Notch1 was higher in lymph node metastases positive cases (χ(2) =7.30, P<0.01). Moreover, higher expression of Jagged1 was found in lymph node positive cases (χ(2) =10.82, P<0.01). CONCLUSIONS: The key protein expression in TGFß1-Smad3-Jagged1-Notch1-Slug signaling pathway significantly correlated with the clinicopathological features of TSCC patients. It's potential as a biomarker and a novel therapeutic target for TSCC patients at risk of metastasis. It may play an irreplaceable role in TSCC progression which may attribute to promoting EMT which enhances cell migration, invasion and metastasis.

MiR-195 Suppresses Cervical Cancer Migration and Invasion Through Targeting Smad3.

OBJECTIVE: MicroRNAs (miRNAs) play crucial roles in cervical cancer development and progression. The purposes of this study were to investigate the role of miR-195 in cervical cancer and clarify the regulation of Smad3 by miR-195. METHODS: Quantitative real-time polymerase chain reaction was used to examine miR-195 expression in cervical cancer tissues and cell lines. The clinicopathological significance of miR-195 down-regulation was further analyzed. Transwell migration and invasion assays were performed. A luciferase reporter assay was conducted to confirm the target gene of miR-195, and the results were validated in cervical cancer tissues and cell lines. RESULTS: MiR-195 was significantly decreased in clinical tissues and cervical cancer cell lines. The low miR-195 level was significantly correlated with higher International Federation of Gynecology and Obstetrics stage, node metastasis, and deep stromal invasion. Up-regulation of miR-195 suppressed cell migration and invasion in vitro. Smad3 was verified as a direct target of miR-195, which was further confirmed by the inverse expression of miR-195 and Smad3 in patients' specimens. CONCLUSIONS: The newly identified miR-195/Smad3 pathway provides an insight into cervical cancer metastasis and may represent a novel therapeutic target.